Antibodies against human herpesvirus-6(HHV-6) and method of use

ABSTRACT

A new human B lymphotropic virus, also designated human herpesvirus-6, has been isolated. DNA, molecular clones, antigenic viral proteins and antibodies having specificity to the new virus have been prepared. Various utilities of the new virus and products derived therefrom have been described.

This is a Division of application Ser. No. 08/392,674, filed Feb. 22,1995 (now U.S. Pat. No. 5,604,093), which was a continuation of Ser. No.07/754,220, filed Aug. 27, 1991 (now abandoned), which was acontinuation of Ser. No. 07/255,712, filed Oct. 11, 1988 (nowabandoned), which was a CIP of Ser. No. 07/228,550, filed Aug. 4, 1988(now abandoned), which was a CIP of Ser. No. 06/901,602, filed Aug. 29,1986 (now abandoned), which was a CIP of Ser. No. 06/892,423, filed Aug.4, 1986 (now abandoned), which was a CIP of Ser. No. 06/895,857 filedAug. 12, 1986, (now abandoned), which was a CIP of Ser. No. 06/895,463,filed Aug. 11, 1986, the disclosure of which is incorporated byreference.

The present invention is related generally to the isolation andcharacterization of a new virus. More particularly, the presentinvention is related to providing a biologically pure, isolated human Blymphotropic virus, molecular clones, nucleic acid, distinctiveantigenic proteins and a method for detecting antibodies to the newvirus. A virus of the type as described herein has not heretofore beenknown or characterized. The nature, properties, importance and variousutilities of the new virus are now presented.

A virus, designated as human B-lymphotropic virus (HBLV or HHV-6 forhuman herpesvirus-6), was isolated from the peripheral blood lymphocytesof six individuals: one HTLV-III(HIV-1) seropositive patient withAIDS-related syndrome, 1 HTLV-III seropositive patient withangio-immunoblastic lymphadenopathy, 1 patient with dermatopathiclymphadenopathy, a patient with Mycosis fungoides, a patient withimmunoblastic lymphoma, and 1 patient(GS) with acute lymphoblastoidleukemia (Table 1). All six isolates were closely related by antigenicand molecular analysis, and sera from all 6 virus positive patientsreacted immunologically with each virus isolate (Table 1). In contrast,only 4 sera from more than 200 randomly selected healthy donors wereseropositive. Subsequent tests showed a high number of normal blooddonors had titers to HHV-6 (59.5%). It was found that HBLV contains alarge double-stranded DNA genome, and is morphologically similar to somemembers of the human herpesvirus group. A detailed morphologicalanalysis of HBLV is given below.

It selectively infects freshly isolated human umbilical cord bloodlymphocytes, B-cells and T cells, where it induces the appearance ofcharacteristic large, retractile mononucleated or binucleated cellscontaining nuclear and cytoplasmic inclusion. bodies. HBLV isdistinguishable from all known human and sub-human primate herpesvirusesby host range, biological effect on infected cells, and by a lack ofimmunologic, antigenic and genomic relatedness (Tables 2 and 3).

Despite morphological similarities, the host range of HBLV was differentfrom all other members of the human herpesvirus group. For example,initial attempts to transmit the virus to a number of T and Blymphoblastoid cell lines, and to a variety of other cell types, wereunsuccessful, but later tests showed that B- and T-cells, megakaryocytesand neural cells could be infected with HBLV. In contrast, Epstein-Barrvirus (EBV) infects most B cells and some epithelial cells. Furthermore,other herpesviruses [e.g., cytomegalovirus (CMV), Herpes Simplex I andII (HSV) and Varicella-Zoster virus (VZV), infect a variety of celltypes, often inducing cytopathic effects. Immunological comparisons withEBV further emphasized these differences. For example, no EBV nuclearantigens were detected in HBLV-infected cord blood mononuclear cells.

The virus of the present invention has been designated humanB-lymphotropic virus (HBLV) because the virus was initially culturedfrom B-cells (the cells had cytoplasmic immunoglobulins) because thevirus initially infects B-cells in vitro in cord blood cultures andbecause HBLV DNA sequences were found in only 3 lymphomas and all 3 wereof B-cell origin. Comparative morphological features which distinguishHBLV from other human herpesviruses are listed in Table 4.

For the identification and isolation of HBLV, fresh peripheral bloodmononuclear cells from AIDS patients with associated lymphoproliferativedisorders were established in cell culture (Table 1). In the cultures ofeight patients, primary cell cultures contained a small number of large,refractile mononucleated or binucleated cells which survive for shortperiods of time. These cells frequently contained intranuclear and/orintracytoplasmic inclusion bodies. Electron microscope examinationrevealed that these cells were infected by a DNA virus, 200 nm indiameter (FIG. 3). These large cells were also the only ones in cultureexpressing viral antigens, as measured by fixed and unfixed cellindirect immunofluorescence assays (IFA) (FIG. 2) and by in situhybridization (FIG. 1). All three virus-positive patients werehomosexual males (2 white and 1 black, between the ages of 35 and 44),who were seropositive for HTLV-III with AIDS-pneumocystic pneumonia,with Kaposi's sarcoma, and with undifferentiated B-cell lymphoma.

The presence of the unique large, refractile cells suggested the needfor further examination of patients demonstrating morphologicallysimilar cells in fresh tissues or culture.

HBLV from all six patients could be transmitted to freshly isolatedhuman leukocytes from umbilical cord blood, adult peripheral blood, bonemarrow, and spleen (previously stimulated with PHA-Pphytohemagglutinin-purified)). After in vitro infection the largerefractile cells, noted in primary cultures, appeared within 2-4 dayspost infection. These cells eventually became the predominant cells inthe culture, surviving for an additional 8-12 days. During this time theother cells in the culture rapidly died. As in primary cell cultures,these large cells expressed viral nucleic acids as shown by in situhybridization (FIG. 1), and viral antigens as detected by IFA(immunofluorescent antibodies), (FIG. 2). Virus production was confirmedby electron microscopy (FIG. 3). HBLV-infected cells were typed forsurface markers defined by specific monoclonal antibodies.

Molecular probes which were derived from HSV-1 (cross reactive withHSV-2), CMV, EBV and VZV were used for comparisons with HBLV. While eachindividual viral probe hybridized to its homologous nucleic acids, HBLVwas clearly distinct from these human herpesviruses (FIG. 10).Furthermore, the size of the HBLV genome was shown to contain a minimumcomplexity of 110 kb-pair as determined by analysis of sucrose gradientpurified viral DNA. Finer analysis indicates the genomic size to beabout 170 kb. This genome size, as well as other features (such asmorphology), also distinguished HBLV from DNA viruses of the adenovirus,polyomavirus, papovavirus, and papillomavirus groups.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of in situ hybridization of HBLV-infected humancord blood cells using pZVH14 HBLV probe.

FIG. 2A is an immunofluorescent analysis of HBLV-infected acetone fixedcells; FIG. 2B shows HBLV-infected live cells expressing membranefluorescence using HBLV antibody; and FIG. 2C shows immunofluorescenceof HBLV-infected cells with serum lacking HBLV antibody.

FIG. 3 is an electron micrograph of HBLV showing extracellular envelopedparticles, the insert represents a virus particl showing envelope,spikes, core, capsid and tegument.

FIGS. 4A, B, and C are electron micrograph of HBLV (negatively stained).

FIGS. 5A and B are Southern blot analysis of HBLV genomic DNA, lanes 1and 2 are positive for HBLV and lane 3 is negative FIG. 5A: Hind IIIdigested HBLV genomic DNA. FIG. 5B: EcoRI digested HBLV genomic DNA.

FIG. 6 shows HBLV proteins after radioimmunoprecipitation using apositive patient (GS) serum and two dimensional (2D) gelelectrophoresis. HBLV specific proteins are indicated by arrowsaccording to apparent molecular size in KDa.

FIGS. 7A and B show the one dimensional (1D) gel electrophoresispatterns of proteins recognized by human and rabbit anti-HBLV serum byradioimmunoprecipitation FIG. 7A: 3 hrs ³⁵ S Cysteine labeled HSB-2infected cells. FIG. 7B: Identification of 120 kd protein using HBLVpositive serum.

FIG. 8 shows restriction enzyme hap of HBLV clone pZVH14.

FIGS. 9A and B show Western blot analyses of HBLV proteins FIG. 9A:Concentrated HBLV from HSB 2 cells. FIG. 9B: HSB 2-Cell Lysates.

FIG. 10 shows dot blot analysis of various herpesviruses, showingspecificity for the probes to their genomic DNA.

FIGS. 11A, B, and C show Southern blots using pZVH14 probe for detectingHBLV in three human B-cell tumors FIG. 11A: HBLV sequences in afollicular large cell lymphoma. FIG. 11B: Detection of HBLV sequences inan African Burkitt tumor. FIG. 11C: Detection of HBLV sequences inMulticentric Tumors arising in a Sjogren's Syndrome patient.

FIG. 12 shows restriction enzyme bands generated using Eco R1 and BamH1as visualized on a 0.8% agarose gel using ethidium bromide staining.

FIGS. 13A and B show restriction endonuclease comparison of a HBLVisolate (HBLV Z29) obtained from the Center for Disease Control and theprototype isolate HBLV(GS). Arrows show the restriction enzymedifferences in the EcoR1 restriction patterns between the two isolatesFIG. 13A Ethidium Bromide straining. FIG. 13B: Hybridization of HHV6Z29and HHV6 HBLV to HBLV probe pzVH14.

FIG. 14 shows the silver stained gel with enriched HBLV proteins.

FIG. 15 is a Western blot of the gel run in parallel with gel of FIG.14. Clearly 120 and 72 KDa proteins are detected.

FIG. 16 is a map of HBLV clone pZVB70.

FIG. 17 shows HBLV infected human umbilical cord blood lymphocytes.Large refractile infected cells are prominent.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned hereunderare incorporated herein by reference. Unless mentioned otherwise, thetechniques employed herein are standard methodologies well known to oneof ordinary skill in the art.

The term "substantially pure" as used herein means that the product isas pure as can be obtained by standard methodology conventional in theart.

Despite morphological and other properties similar to some of theherpesviruses, human B lymphotropic virus (HBLV) is a new humanherpesvirus. It is distinguishable from other viruses by biologicalproperties and by the lack of immunological and genomic homology. HBLVis highly lytic in vitro, as are CMV, HSV, HVS (Herpesvirus Simia), andHVA (Herpesvirus ateles), but has a different host range than theseviruses. It is possible that HBLV could indirectly cause abnormalitiesin B-cells leading to malignancy in vivo.

Even though in certain instances HBLV was associated with humanT-lymphotropic virus-III/lymphadenopathy-associated virus (HTLV-III/LAV)seropositive donors, other evidence indicates that it is not exclusivelyan AIDS-associated agent. Not only did all HTLV-III seropositivepatients have complicating lymphoproliferative disorders, but HBLV wasalso isolated from a HTLV-III seronegative ALL (acute lymphocyticleukemia) patient. Furthermore, some seroepidemiological analyses haveshown a reactivity clearly dissociated from HTLV-III antibody positiveindividuals.

Serological comparisons demonstrate the uniqueness of HBLV.Immunofluorescence assay was developed following techniques originallydescribed for herpesviruses, and was used to analyze patients andhealthy control sera, and to monitor infected cells. Sera from all sixHBLV positive patients demonstrated an IgG antibody titer to viralcapsid antigens (>1:20). In contrast, only 4 of the more than 200 serafrom randomly selected healthy donors were positive. Subsequentserological surveys indicate the prevalence of HBLV antibodies in normalpopulation to range from about 9% to about 47% with regionaldifferences. The pattern of immunofluorescent staining in fixed,infected cells varied from punctate nuclear staining to diffuse stainingof the entire cell (FIG. 2A). In live cells, the staining was confinedto the cell membrane either as a partial ring or in a capped form (FIG.2B and C). Uninfected cord blood mononuclear cells were negative whentested with Sera from the 6 HBLV positive patients. Sera from thesepositive patients also contained antibody to EBV and CMV. A carefulcomparison of the titers of antibody to EBV, CMV, and HBLV yielded adistinct titer for HBLV as compared to that for EBV and CMV.Furthermore, the reactivity to EBV, CMV, HSV-1 and 2 and VZV wascompletely removed by adsorption with disrupted, EBV-infected cells orwith purified viruses, without significantly affecting the antibodytiter to HBLV.

Sucrose gradient purification of HBLV. Heparinized peripheral bloodleukocytes or human umbilical cord blood mononuclear cells are banded inFicoll-Hypaque and established in cell culture at 36° C. following PHA-P(5 ug/ml) stimulation for 48 hours. The cells are then grown inRPMI-1640 medium supplemented with 10% fetal bovine serum (heatinactivated, 56° C. for 30 min.) and 5 ug/ml hydrocortisone. Frozensupernatants obtained from the infected cells are thawed, collected in250 ml tubes and spun at 3500 rpm in a Sorvall GSA rotor at 5° C. for 10min. The clarified supernatants are transferred to SW28 tubes and spunand pelleted at 17,000 rpm for 90 min. at 5° C. Pellets obtained areresuspended in 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 1 mM EDTA (TNE) to avolume of 300 microliters and layered onto a 15-60% sucrose gradient andspun in an SW41 rotor (Beckman) at 20,000 rpm for 30 min. at 5° C.Fractions of 1 ml are collected from the top of the gradient. Eachfraction is diluted to 10 ml, spun, and pelleted in an SW41 rotor at17,000 rpm for 90 min. Pellets are resuspended in 300 microliters of TNEand aliquots assayed (by ELISA and Western Blot) for the presence ofvirus and for virus infectivity. Human B Lymphotropic Virus is easilydetected in fractions 4-9 with a peak in fractions 5-7 by both assays.Extraction of nucleic acids from each fraction shows the presence ofdouble stranded DNA in fractions 5-9 with a peak in fraction 7. Virus isalso detected by electron microscopy in the SW41 gradient pellet aswell. Virus purified from fresh unfrozen supernatants according to thisprocedure is used for detailed electron microscopy.

Aliquots of the sucrose gradient fractions can be definitively assayedfor the presence of HBLV by DNA dot blot analysis using the pZVH14 9 kbinsert (FIG. 8) as a probe. The pZVH14 molecular clone is obtainablefrom the American Type Culture Collection under Accession No. 40247.

The immunofluorescence, Western blot and radioimmuno-precipitationassays are also employed for detecting HBLV infection and HBLVantibodies in a variety of hematopoietic malignancies, including B-celllymphomas of both AIDS and non-AIDS origin. The presence of HBLVantibodies is elevated in the following disease groups, but theinvention is not intended to be limited to these specific diseases:

Roseola (Exanthum subitum)

Burkitt's lymphoma

Hodgkin's disease

Mononucleosis-like syndromes

Sarcoidosis

Sjogren's Syndrome

A newly described infectious disease syndrome similar to that seen inLake Tahoe characterized as an "acute mononucleosis-like syndrome" inadults, commonly known as chronic fatigue syndrome (CFS).

ALL (acute lymphocytic leukemia) as diagnosed in children of Japanese,Caribbean and African origin.

HIV-1 antibody positive AIDS, ARC and PGL (persistent generalizedlymphadenopathy) patients.

HBLV Virus Propagation. Infection of human umbilical cord blood orperipheral blood mononuclear cells is conducted by cell-freetransmission as follows:

1) Fresh blood samples are diluted 1:1 with RPMI-1640 and spun (andbanded) on a Ficoll gradient.

2) The banded mononuclear cells are washed and put into culture in thepresence of PHA-P (5 ug/ml) and hydrocortisone (HC) (5 ug/ml) in 20%fetal calf serum (FCS) and RPMI-1640.

3) After 24 hours, polybrene (2 ug/ml) is added to the culture and after6-24 hours, the cells are pelleted.

4) A one ml aliquot of freshly harvested or frozen infected culturesupernatant is added to the pellet and incubated at 37° C. for 1-2 hour,with frequent agitation.

5) Fresh medium [10% FCS and HC (5 ug/ml) in RPMI-1640] is then added tothe suspension, cultured, and incubated at 36° C.

6) Within 2-10 days post infection, the characteristic enlargedrefractile cells become visible. Supernatant is harvested at the peak ofinfection as measured by immunofluorescence and by visual observation ofthe culture for further transmission.

Cells infected by HBLV were also used to directly compare immunologicalcross-reactivities with other human and nonhuman primate herpesvirusesusing specific monoclonal antibodies, hyperimmune sera, or sera fromantibody positive control donors. As summarized in Tables 2 and 3,monoclonal antibodies to EBV, CMV, HSV, and hyperimmune sera to RhesusCMV and African Green CMV, did not react with HBLV-infected cells. Humansera possessing anti-bodies to EBV, CMV, HSV, and VZV also did not reactwith HBLV-infected cells. Furthermore, sera from several Old World andNew World primates, many of which had antibodies to nonhuman primateherpesvirus (including EBV-like viruses and CMV), did not show anycross-reactivity with HBLV-infected cells (Table 2).

Immunofluorescent Analysis of HBLV-infected cells. A modification of theindirect immunofluorescence assay developed by Henle et al (J.Bacteriol. 91:1248-1256) for EBV was used for the detection of antibodyto HBLV capsid antigens. For this assay, HBLV-infected cord bloodmononuclear cells were isolated by Ficoll gradients to remove deadcells. Uninfected human cord blood mononuclear cells were used ascontrols. Uninfected and infected cells were washed 3 times for 10minutes with PBS without Mg++Ca++, resuspended in PBS containingMg++Ca++, deposited on TEFLON coated slides, air dried, and fixed incold acetone for 10 minutes. Patient's sera (heat inactivated at 56° C.for 30 minutes and clarified by centrifugation) were added to theacetone fixed cells, incubated in a humidity chamber at 37° for 40minutes, washed with PBS, air dried, and stained with affinity purifiedFITC conjugated anti-human IgG (H and L) for 40 minutes. The cells werecounterstained with Evans blue (1:500 dilution in PBS) for 5 min tofurther reduce background due to autofluorescence. The cells were againwashed as above, air dried, and mounted with IFA iummunofluorescenceassay). mounting solution. Large cells with greenish to yellow granularimmunofluorescent and cytoplasmic staining were scored as positive cellsfor HBLV. The example of assays carried out 5 days post infection areshown in FIG. 2a. Small cells in the background did not react withpatient serum (FIG. 2b with arrows).

As is shown in FIG. 2, detection of viral membrane antigen HBLV infectedas well as uninfected live cells (non-fixed) were washed 3 times inserum-free RPMI1640 medium and treated with patient's serum for 30minutes at 4° C. The cells were again washed, treated with affinitypurified FITC anti-human IgG for another 30 minutes, washed in mediumagain and examined for membrane fluorescence. HBLV infected cells showedsurface markers when tested with patient serum using theimmunofluorescence technique (FIG. 2b).

Southern blot analysis of HBLV genomic DNA. Supernatant fluid from HBLVinfected umbilical cord blood cells was layered onto 20% glycerolcushions and pelleted by centrifuging at 25,000 rpm for 3 hr. in aBeckman SW41 rotor at 4° C. The pellets were suspended in TNE buffer (10mM, Tris-HCl, pH 9; 100 mM, NaCl; 1 mM EDTA), and extracted with PCI9(Phenol:Chloroform:Isoamyl alcohol; 50 mM Tris-HCl, pH9;100:100:1:10::v:v:v:v) followed by Chloroform:isoamyl alcohol(24:1::v:v). Substantially enriched viral DNA was precipitated by adding2 volumes of 95% ethanol. DNA was digested with Hind III and cloned intothe Bluescribe vector (commercially available from Vector CloningSystems, Calif.). Several clones obtained were prepared as radiolabeledprobes and screened for specificity of hybridization by Southernblotting to HBLV infected human umbilical cord blood cell DNA and by insitu hybridization to such infected cells. Results of hybridization ofHBLV clone pHV14 to DNA from pelleted virus digested with Hind III andEcoR1 are shown in FIG. 5. Extracellular virus is shown in lane 1, virusinfected human umbilical cord blood cells in lane 2 and negative controlDNA isolated from the skin of an AIDS patient in lane 3. Clone pZVH14scored positive in these assays and did not hybridize to uninfectedcontrols. The infected cell DNA shown in lane 2 is isolated insubstantially pure form after several rounds of cell free virustransmission in human umbilical cord blood cells.

In addition to the procedures described above, the following specificmethods and materials may also be employed.

Rather than using cord blood cells, HBLV can also be propagated byinfecting other suitable host cells such as HSB2 cells obtainable fromATCC (CCl 120.1). HBLV(GS) strain was collected from 15 liter culturesof infected HSB2 cells by continuous flow centrifugation onto 10% to 60%sucrose gradients. Bands collected between 1.135 and 1.210 g/ml werepelleted at 20,000 rpm and resuspended in PBS containing 1 mMphenylmethylsufonyl fluoride (PMSF) and 10 mM MgCl₂. The suspendedvirions were subjected to six strokes in a Dounce homogenizer and 23units per ml of RNAse free DNAse (Boehringer-Mannheim) and incubated for10 min. at 37° C. The total volume (2 ml) was layered onto 36 ml 5-30%dextran T10 gradient (w/w) in 0.5 mM phosphate buffer, pH7, andcentrifuged in a Beckman SW 27 rotor for 1 hr. at 20,000 rpm at 4° C.(Dolyniuk et al, J. Virol. 17:935, 1976). Fractions of 4 ml werecollected and a visible band was collected in fractions 7-9. Examinationof fraction 10 under the electron microscope revealed highly enrichedvirions with very little cellular debris. Electron microscopeexamination of virions filtered through 0.2% polyvinylpyrollidone (PVP)treated 0.45 um Nalgene fitters also gave excellent results and proteingel analyses showed a purification indistinguishable from fractions 7-9above by electron microscopy (FIG. 3).

Purification of HBLV Genomic DNA

Infection and banding of the virions by continuous flow centrifugationwas as described herein supra. The sucrose-banded virus was pelleted at20,000 rpm in a Beckman SW27 rotor for 90 min. The virus was resuspendedin 400 ul of TE buffer (20 mM Tris-HCl, 1 mM EDTA) and 130 ul of 10%sodium lauryl sarkosinate added. The viral lysate was incubated at ₆₀°C. for 20 min. and then layered onto a 54% CsCl, 0.1 mg/ml ethidiumbromide solution and centrifuged in a Beckman SW50 at 45,000 rpm for 20hr. at 20° C. The Viral DNA band (1/3 from the top of the gradient) wasvisualized under UV illumination and removed by side puncture with aneedle and syringe. The HBLV DNA-CsCl aliquot was extracted 5 times withequal volume of n-butanol and then dialyzed against 2 changes of 1000 mlof TE buffer at 4° C. Dialysis membrane was placed over an Eppendorftube and held in place with an Eppendorf cap into which a hole had beenbored. The tube was inverted and floated on the buffer for dialysis. DNAprepared in this way was substantially pure to visualize theethidium-stained restriction digests on agarose gels and for thecreation of plasmid vector libraries. The DNA yield is usually greaterthan 30 ug per 25 liters of cell free supernatant depending on theextent of the infection.

Labeling of Cells

Media for 24 hr. labeling incubations was prepared by mixing 8 ml ofmethionine Free DMEM (D-Met) (Gibco), 2 ml of 50% fetal calf serum inRPMI 1640 and 0.1 ml gentamicin (100×concentrated, 5 mg/ml). Media for2-3 hr. labeling incubations contained D-Met and 10% fetal calf serum.The amount of 5 mCi of [³⁵ S] methionine (or other radiolabeled aminoacid) was lyophilized and reconstituted with 400 ul of D-Met. Cells inthe amount of 5×10 were pelleted at 1000 rpm for 5 min. in the SorvallGLC bench top centrifuge and resuspended in labeling media. For 24 hr.labeling, the cells were split into two 0.8 ml aliquots in a 24 wellmicrotiter plate and 50 ul of the reconstituted [³⁵ S] methionine wasadded to each. For 2-3 hour labeling, 5×10 cells were resuspended in 1.0ml of labeling medium and split into two 0.5 ml aliquots and 50 ul ofradiolabeled methionine added to each. Cells were incubated at 37° under5% CO₂ and 85% humidity for the period of time necessary for labeling.

Radioimmunoprecipitatior

After metabolic labeling, (as described herein supra) the cells werediluted in 10 ml of ice-cold phosphate buffered saline (PBS) andpelleted for 5 min. at 1000 rpm in the Sorvall GLC bench top centrifuge,resuspended in 10 ml of fresh ice-cold PBS and pelleted a second time.The cells were resuspended in 1 ml of PBS and transferred to anEppendorf tube and centrifuged at half maximal speed for 2 min. About550 ul of lysis buffer [0.1% SDS (sodium dodecyl sulfate), 1% TRITONX-100, (T-octylphenoxypolyethoxyethanol, Sigma Chemical Company, St.Louis, Mo.) X-100, 1% desoxycholate (free acid), 20 mM Tris-HCl, pH 8.0,150 mM NaCl and 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma)] wasadded. The lysate was vortexed at a setting of 5 for 15 sec., allowed tosit on ice for 0 min. and vortexed again. The samples were thencentrifuged at top speed in an Eppendorf centrifuge for 3 min. A 50 ulstock aliquot was removed from each tube and immediately frozen on dryice. The remaining supernatant was transferred to a clean Eppendorf tubeand 20 ul of sera was added. The tubes were placed on a rotor at 4° C.and gently inverted for 12 hr. The samples were then centrifuged at topspeed for 2 min. and all but 10 ul of the supernatant was removed to anew Eppendorf tube. The amount of 100 ul of a 50% (v/v) slurry ofprotein A SEPHAROSE, beaded agarose (Pharmacia) in lysis buffer wasadded to each tube and the tubes gently inverted for 30 min. The sampleswere centrifuged for 2 min. at top speed and the supernatants discarded.The protein A SEPHAROSE, beaded agarose pellet was washed 6 times byresuspension in lysis buffer and centrifuged for 15 sec. at top speed.After removal of the supernatant of the sixth wash, the pellet wasfrozen and sent to Protein Data Bases, Inc. (a commercial analyticalservice laboratory in Huntington Station, N.Y.) for the gel runs.

All radioimmunoprecipitations were performed using serum from patientGS, the source of the prototype HHV-6 isolate. Specificity of theantisera was demonstrated by adsorbing the sera against virionpreparations of human cytomegalovirus, Epstein-Barr virus, VaricellaZoster virus, and Herpes Simplex type 1.

High Resolution 2 Dimensional Gels (HR2D) of HHV-6 Proteins

The viral proteins were prepared by SDS-BME (sodium dodecylsulfate-basic maintenance emulsion) lysis of gradient-banded virions andRNA-DNAse treatment as described herein supra and then frozen on dry iceaccording to the standard protocols of Protein Data Bases Incorporated(PDI), Huntington Station, N.Y. The samples were run at PDI on 12.5%broad range non-equilibrium and equilibrium polyacrylamide gels andsilverstained. The protein-A Sepharose bound radiolabeledimmunoprecipitates run on 12.5% broad range non-equilibriumpolyacrylamide gels were then exposed for autoradiography at PDI.

It should be noted that in addition to radioimmunoprecipitation (RIP),Western blot, indirect immunofluorescence assay (IFA) enzyme linkedimmunosorbent assay (ELISA), and the like can also be utilized to detectviral antigens or antibodies. These techniques are well established andknown to one of ordinary skill in the art to which this inventionbelongs.

HR2D Western Blotting

Immunoblotting was performed after HR2D electrophoretic resolution offractions of HBLV prepared from sucrose gradients or filtered virus asdescribed herein supra. The nitrocellulose sheets were stored at 20° C.prior to use. Sheets were incubated for one hour in a blotte solution of4% normal goat serum, 4% fetal bovine serum, 5% non-fat dry milk and0.02% thimerosal for blocking. Sheets were then incubated with serumfrom a known HBLV infected patient, diluted 1:1000 in the blottosolution. After 3 successive 5 minute washes with PBS, they were reactedin sequence for 1 hour with 1:500 dilution of affinity purified goatanti-human IgG labeled with biotin and for one hour with 1:1000 ofhorseradish peroxidase streptavidin (Kirkegaard and Perry Labs., Inc.,Gaithersburg, Md.) at room temperature (about 21°-25° C.) in 5% normalgoat serum in PBS and 0.02% Thimersol. A stock solution of4-chloronaphthol (4CN stock) was prepared by dissolving 0.3 g of4-chloronaphthol in 100 ml of methanol. Staining was carried out in asolution containing 2 ml of 4CN stock, 8 ml of PBS and 4 microliters ofhydrogen peroxide. The reaction was stopped by washing with distilledwater.

In order to obtain a better size estimate, DNA was purified from viruscollected by continuous flow centrifugation from 15 liters of HBLVinfected HSB2 cell culture supernatant and pooled. The regions of the10% to 60% sucrose gradient pooled were from 1.14 to 1.17 g/ml, fractionA, and from 1.17 to 1.21, fraction B. The virions were pelleted, lysedand the DNA purified by banding on cesium chloride gradients anddialyzed. FIG. 12 shows the restriction enzyme bands generated usingEcoRI and BamHI as visualized on 0.8% agarose gels by ethidium bromidestaining. Over 26 bands were generated by EcoRI digestion (A to Z, topto bottom) and at least 15 fragments with BamHI (A to O). The bands seenwere of similar intensity with a marked absence of submolar fragments.compared to other herpesviruses. Possible exceptions were the EcoRI A'and the BamHI F' and M' fragments which had intensities equivalent to1/4 M. The reasons for the generation of these bands are not understood;however, they are possibly due to genomic inversions and were notcounted for the genome size estimates. Table 5 shows the results ofrestriction enzyme analyses of HBLV.

The construction of BamHI plasmid libraries from the DNA showed thatnearly 100% of the fragments cloned were HBLV thereby providing furtherevidence that the bands visualized in FIG. 12 can be used as a reliableestimate of the HBLV genome size. The molecular weights of the fragmentslisted in Table 5 gave genome size estimates of 168,000 and 172,000 forthe EcoRI and BamHI digests, respectively. By this estimate, the genomeof HBLV is approximately the size of the Epstein-Barr virus genome.

Restriction endonuclease comparison of another independent HBLV isolate,HBLV(Z29), to the prototype HBLV(GS) strain is shown in FIG. 13. Thearrows indicate the areas where the EcoRI digests of each stain differas visualized by ethidium bromide staining. Hybridization to one HBLVprobe, ZVH14, revealed identical restriction patterns between the twoisolates; however, by probe ZVB70, the HBLV(GS) BamHI B fragment, showeddifferences (not shown). This indicates that restriction siteheterogeneity can be observed among different isolates of HHV-6. Anotherisolate, HBLV(DV), was identical by hybridization with both the ZVH14and ZVB70 probes to HBLV (GS).

Studies of the complexity of the enveloped HHV-6 proteins were attemptedby banding the virus collected by continuous flow centrifugation onDEXTRAN T-10 gradients similar to methods used to purify the proteins ofthe enveloped EBV (Dolyniuk et al, 1976, supra). The virions obtainedfrom continuous flow centrifugation were pelleted, treated with DNAse 1and then banded on 10% to 30% DEXTRAN T-10 gradients. The variousfractions collected from the top were analyzed by electron microscopyand the virus was pelleted from those which looked relatively free ofcellular debris. A viral band was seen toward the bottom of the gradient(fractions 7-9) and the fraction immediately below (fraction 10) wasconsidered to be relatively free of cellular debris when compared tovirus obtained after a single banding. The virus was found in clusterswith little cellular material. Virus prepared by this method wheninoculated into rabbits resulted in the generation of HBLV specificantibodies in 14 days which were readily detected by indirectimmunofluorescence assay on infected cells. Subsequent bleeds gave somenon-specific cellular background in IFA tests. Hence, the animals shouldbe bled about 14 days post inoculum. These antibodies can be utilizedfor detection of HBLV by established techniques.

The pure preparations of the virus as revealed by electron micrographs,were used to determine the proteins by direct visualization on highresolution two dimensional polyacrylamide gels (HR2D). As mentionedherein supra by procedures developed at Protein Data Bases, Inc.,several 12.5% broad range non-equilibrium. gels were run and a gel ofthe virions obtained from the DEXTRAN T-10 fraction 10 was silverstained(FIG. 14). A parallel gel was run and Western blotted using GS serum andperoxidase conjugated goat anti-human antibody. Two major proteins weredetected at 120 and 72 kDa as shown in FIG. 15. Radioimmunoprecipitationof HBLV-infected cell lysates with the GS serum showed severaladditional proteins (FIG. 6). In addition to the proteins detected byWestern blots, radioimmunoprecipitations performed on proteins fromlysates of metabolically labeled cells showed a protein at 120 kDa:however, additional major proteins at 200, 80 and 19 kDa, as well assome minor proteins at 60 kDa, 80 kDa and several in the 30 kDa rangewere also detected. Two forms of the 19 kDa proteins were observed, amore acidic form, 19a, and a more basic major form, 19b, possibly due todifferences in phosphorylation (FIG. 6). The antigenic proteins can thenbe isolated in substantially pure form following standard purificationtechniques, such as column chromatography, HPLC, preparative gelelectrophoresis, and the like. These proteins can be identified, forexample by Western blot using HBLV antibody positive sera. Apharmaceutical composition in accordance with the present inventioncomprises an immunogenic amount of the antigenic protein in apharmaceutically acceptable carrier. Antigenic proteins or portionsthereof can also be obtained from gt11 expression libraries or the like.

Antigenic proteins of the present invention also allow detection of thepresence of HBLV antibodies in a biological sample by reacting saidsample with the viral antigens, a positive. antigen-antibody complexformation being indicative of HBLV infection. Antigen-antibody reactionscan be detected by any standard immunological techniques well known toone of ordinary skill in the art, such as radiommuno, Western blot,ELISA, immunofluorescence, histoimmunological tests and the like.

EXAMPLES Example 1

Fresh tissue sections from 3 patients were found to contain a low numberof HBLV-infected cells. One patient, a 40 year old Hispanic with ahistory of IV drug use, was seropositive for both HTLV-I and HTLV-III,and was diagnosed with AIDS-pneumocystic pneumonia with associateddermatopathic lymphadenopathy. Another was a 61 year old white male whoreceived multiple blood transfusions in conjunction with open heartsurgery 4 years prior to death. This patient was seropositive forHTLV-III and was diagnosed with immunoblastic lymphadenopathy with someskin involvement. A third patient (GS) was a 16 year old black malediagnosed with acute lymphocytic leukemia of the T-cell type. Unlike theothers, this patient was seronegative for HTLV-III. Primary peripheralblood mononuclear cell cultures from these patients also contained asmall number of the unique cells which, upon close examination, werealso found to be infected by HBLV.

Example 2

A direct comparison of molecularly cloned sequences of the HBLV genomewith the genomes of other herpesviruses was also conducted. Several DNAclones obtained from nucleic acids extracted from purified virus wereexamined for specificity and for comparison with other DNA viruses. TwoHBLV clones, designated. pZVH14 (FIG. 8) and pZVB70 (FIG. 16, ATCC No.40473), were used in these studies. Southern blot analysis (FIG. 5)showed the presence of viral specific DNA in Hind III and EcoRI digestsof DNA from both purified virus and HBLV-infected human cord bloodcells. In situ hybridization experiments with the pzVH14 probe alsoconfirmed that these sequences were confined to the infected cells (FIG.1).

Example 3

Monoclonal antibodies and hyperimmune sera prepared against human andsimian herpesviruses were tested for reactivity with HBLV infected cellsby indirect immunofluorescence procedures as described herein above.Monoclonal antibodies to EBV and HCMV were used at 1:40 dilution; HSV-Iand II, VZV and HVS at a 1:10 dilution and normal ascites fluid was usedat 1:5 and 1:10 dilutions. Hyperimmune sera to African green and Rhesusmonkey CMV were heat inactivated (50° C. 30 min.), clarified at 10,000rpm, and then were used at 1:10 dilutions. In addition to the serashown, human sera containing antibodies to EBV, CMV, HSV-I and II, andVZV also did not react with HBLV infected cells. African green monkeyand Rhesus sera containing antibody to CMV were also negative whentested with HBLV. Monoclonal antibodies to EBV and HCMV, and ascitesfluid from normal mouse were gifts from Dr. Gary Pearson, School ofMedicine, Georgetown University, Washington, D.C. Monoclonal antibodiesto VZV and HVS were obtained from Dr. Nancy Chang, Baylor College ofMedicine, Houston, Tex., and Dr. John Dahlberg, NCI, Bethesda, Maryland,respectively. HSV-I and II monoclonal antibodies were purchased fromDupont, Boston, Mass. Hyperimmune serum to purified African green andRhesus CMV were previously prepared in rabbits by Dr. Ablashi. Thespecificity of the serum containing antibodies to HBLV was shown byadsorbing it against the other human herpesviruses (either whole virusor infected cells).

Abbreviations used: HBLV, Human B lymphotropic virus; EBV, Epstein-Barrvirus; HCMV, Human cytomegalovirus; HSV, Herpes simplex virus; VZV,Varicella-Zoster virus; HVS, Herpes virus saimiri, VCA (Viral capsidantigen); EA, early antigen; MA, membrane antigen.

HBLV infected cord blood mononuclear cells were stained with an HBLVnegative serum resulting in a considerable number of large cells with noimmunofluorescence.

Example 4

Serum from Old World and New World primates were tested for antibody toHBLV by indirect immunofluorescence as described.

Some sera from the Old World primates were gifts from Dr. P. Kanki,Harvard School of Public Health, Boston, Mass. All sera were heatinactivated at 50° C. for 30 minutes, and clarified by centrifugationbefore use. HBLV-infected cord blood leukocytes, P3HR-1 (an establishedcell line expressing EBV-VCA), and Owl monkey kidney cells infected byHSV-strain II were used for comparisons. When infected cells showedcytopathic effects, the cells were fixed in acetone and used for the IFAtest.

Three owl monkeys and one cottontop marmoset were previously inoculatedwith HVS. Sera from these animals possessed antibody to HVS late antigenwhich cross-reacted with Herpesvirus ateles. The results are presentedin Table 2.

Example 5

In situ hybridization of HBLV-infected human cord blood cells. Testswere performed utilizing ³⁵ S-labeled RNA probes as described hereinsupra. Clone pZVH14 of the HBLV genome was used as a template forradiolabeled RNA using T7 RNA polymerase, ³⁵ S-labeled GTP, andunlabeled ribotriphosphates. Less than one grain per cell was observedin uninfected negative control cultures. Large retractile cellscharacteristic of the infected cultures were heavily labeled, indicatingthe expression of abundant viral messages (FIG. 1).

Example 6

Two dimensional gel electrophoresis patterns of proteins recognized byhuman sera against human B cell lymphotropic virus (HBLV) are shown inFIG. 6. Human umbilical cord blood lymphocytes or HSB₂ cells wereinfected with HBLV and then labeled by incubation with ³⁵ S-methioninefor periods of either 3 hours or 24 hours. H9 cells were used asnegative controls. The labeled cells were lysed and the proteinsimmunoprecipitated according to established procedures (Protein DataBases, Inc., New York). Spots seen on the gels of the lysates frominfected cells but not seen on the control gels represent candidatevirus proteins arrayed in unique virus specific patterns. These patternsserve as a fingerprint which can specifically identify HBLV. Theproteins detected are antigenic proteins, the coding sequence of whichcan be cloned and expressed, and the purified proteins thus obtained canbe used as diagnostic reagents.

Preparation of the Clones

Of course, the availability of the biologically pure HBLV and its DNA,allows the preparation of the clones of HBLV. A general method ofcloning the Human B Lymphotropic Virus (HBLV) genome involves isolatingviral DNA after infection of suitable host cells (such as HSB₂ and thelike), primary cells or cord blood cells with the HBLV virus. Theunintegrated viral DNA is then cloned in a suitable cloning vector suchas a plasmid or a lambda phage to create libraries which can be screenedfor the presence of viral specific DNA fragments.

Infected cells and cultured peripheral cord blood cells produce HBLVvirus and serve as the principal source of the virus for immunologicalassays and the like for detecting virus-specific antigens and antibodiesin human sera. Cultures of infected cells are grown and the virusharvested from the supernatant and the high molecular weight DNAextracted from the virus. This produces viral DNA containing the HBLVgenome of the present invention. This DNA is then subcloned in asuitable plasmid to produce a clone. A complete description of theprocedures for preparing clones can be found in such standardpublications as Maniatis et al: "Molecular Cloning," Cold Spring Harbor,N.Y.

Two elements of the above process are well known are a part of therecombinant DNA procedures: the DNA library and the differentialscreening of DNA inserts to infected and uninfected cells. The libraryis formed by taking the total DNA from the enriched or purified virusDNA, cutting the DNA into fragments with suitable restriction enzyme(s),joining the fragments to plasmid vectors, and then introducing therecombinant DNA into a suitable host. The viral specific DNA fragmentsare distinguished by their hybridization to infected cell DNA and/or byin situ hybridization to infected cells but not to uninfected cells.

As shown herein infra, a molecular clone, pZVH14, of the HBLV genome isuseful as a template for radiolabeled RNA using T7 RNA polymerase, ³⁵S-labeled GTP, and unlabeled ribotriphosphates.

In the preferred embodiment of the present invention, supernatant fluidfrom HBLV infected cells is layered onto 20% glycerol cushions andpelleted by centrifuging at 25,000 rpm for 3 hr. in a Beckman SW41 rotorat 4° C. The pellets are suspended in TNE buffer (10 mM, Tris-HCl, pH 9;100 mM, NaCl; 1 mM EDTA), and extracted with PCI9(Phenol:Chloroform:Isoamyl alcohol; 50 mM Tris-HCl, pH9; 100:100:1:10v:v:v:v) followed by chloroform;isoamyl alcohol (24:1::v:v). Enrichedviral DNA is precipitated by adding 2 volumes of 95% ethanol DNA isdigested with Hind III and cloned into the Bluescribe vector(commercially available from Vector Cloning Systems, CA). Several clonesobtained after screening with labeled, enriched, DNA were examined forspecificity of hybridization to the HBLV DNA and by in situhybridization to HBLV infected cells.

Clones pZVH14 and pzZVB70 which were thus produced, scored positive whentested by hybridization techniques and did not hybridize to uninfectedcontrols. The infected cell DNA is isolated after several rounds of cellfree virus transmission in human umbilical cord blood cells or HSB₂cells. Clone pzVB70 was obtained from CsCl gradient banded DNA ofsucrose banded virus. DNA was BamH1 digested as described herein supra.

It is noted that these probes, either alone or in combination, can beemployed for detecting the viral DNA or RNA and virus-infected cellscontaining HBLV nucleic acids by any of several standard techniques wellknown to one of ordinary skill in the art. Examples of such wellestablished techniques are Southern and dot-blot for DNA analysis,Northern blot for RNA analysis and in situ hybridization. Furthermore, aprobe for in situ hybridization can be made by any of well establishedprocedures such as radiolabeling or covalent linkage of hapten or enzymeto DNA. A few illustrative examples are now provided.

Example 7

Several DNA clones obtained from nucleic acids extracted from purifiedvirus obtained as described above, were examined for specificityrelative to other DNA viruses. HBLV clone designated pZVH14, contained a9.0 kb Hind III fragment. Southern blot analysis showed the presence ofviral specific DNA in Hind III and EcoRI digests of DNA from bothpurified virus and HBLV-infected human cord blood cells. In situhybridization tests with the same probe also confirmed that thesesequences were confined to infected cells.

Example 8

Human B Lymphotropic Virus clone pZVH14 has been restriction enzymemapped as shown in FIG. 8.

Example 9

Similarly, HBLV clone pZVB70 has been restriction enzyme mapped as shownin FIG. 16.

It is noted that based on the sequence information, any number ofspecific clones can be generated and used as probes. The techniques arewell established and known to one of ordinary skill in the art to whichthis invention belongs.

Example 10

In situ hybridization of HBLV-infected cells. Tests were conductedutilizing ³⁵ S-labeled RNA probes as described herein supra. ClonepZVH14 of the HBLV genome were used as a template for radiolabeled RNAusing T7 RNA polymerase, ³⁵ S-labeled GTP, and unlabeledribotriphosphates. Less than one grain per cell was observed inuninfected negative control cultures. Large retractile cellscharacteristic of the infected cultures were heavily labeled, indicatingthe expression of abundant viral messages (FIG. 1).

Example 11

Based on the nucleotide sequence, polymerase chain reaction technique(Saiki et al, 1985, BioTechnology, 3:1008; Science, 230:1350) wasemployed to obtain increased levels of nucleic acids from specimens(tissue or cell culture) suspected of HBLV infection from diseased andnormal A (control) populations and the presence of HBLV detected bySouthern blotting of the amplfied HBLV DNA or other method of detectingthe amplified DNA with radiolabeled or nonradiolabeled probes as arewell known to one of ordinary skill in the art.

A deposit of the clones pZVH14 and pZVB70 have been made at the ATCC,Rockville, Md. under the accession numbers 40,247 and 40,473,respectively. The deposit shall be viably maintained, replacing if itbecomes non-viable, for a period of 30 years from the date of thedeposit, or for 5 years from the last date of request for a sample ofthe deposit, whichever is longer, and made available to the publicwithout restriction in accordance with the provisions of the law. TheCommissioner of Patents and Trademarks, upon request, shall have accessto the deposit.

In summary, as demonstrated herein, high level production of HBLV cannow be obtained by the use of the HSB2 or other cell lines. This allowspurification of the enveloped virus and the viral nucleic acids.Purification of the viral DNA has been demonstrated by hybridizationwith specific cloned viral DNA such as clone paVH14. Although the sizeestimate (170,000) of the HBLV genome is similar to that of EBV,evidence by molecular hybridization shows distant relationships to thehuman cytomegalovirus and to the Marek's disease cirrus of chickens(data not shown).

Comparison of the Western blots from the HR2D gels to theradio-immunoprecipitation revealed a major antigenic protein of 120 kDaand other antigenic proteins described herein supra which is detectableby both (RIP and Western blot) methods. The 120 Kd protein seems to be amajor antigenic protein as demonstrated by anti-HBLV patient sera.Increased resolution of the minor proteins on 2D gels indicates that itwould be easier to verify the presence of characteristic viral proteinsby this method than by 1D gels. Very clean background seen in the twodimensional Western blot, in which 120 Kd and 72 Kd proteins weredetected, may be the method of choice.

Although not necessary, because biologically pure virus can be obtainedby following the standard procedures described herein by anyone ofordinary skill in the art, nevertheless a deposit of the isolated virushas been made at the ATCC, Rockville, Md. under accession number VR2225.A deposit of the anti-HBLV positive serum has also been made at the ATCCunder accession number 40476. The deposits shall be viably maintained,replacing if it becomes non-viable, for a period of 30 years from thedate of the deposit, or for 5 years from the last date of request for asample of the deposit, whichever is longer, and made available to thepublic without restriction in accordance with the provisions of the law.The Commissioner of Patents and Trademarks, upon request, shall haveaccess to the deposit.

A diagnostic kit in accordance with the present invention comprisescontainers separately containing anti-HBLV antibodies, one or morepurified or cell associated antigenic viral protein(s) produced by HBLVin any part of its replicative cycle (i.e., HBLV infected cells or cellsexpressing specific HBLV proteins); HBLV specific nucleic acid probes;positive and negative controls and instructional material to performdiagnostic test employing said antibodies, antigenic viral protein(s),probes and the like. Of course, the present invention also allows thedetection of HBLV present in any biological sample. Any suitable methodmentioned herein can be utilized as deemed most appropriate by one ofordinary skill in the art, depending on such factors as the location,nature, amount of the sample available and the like.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

                  TABLE 1                                                         ______________________________________                                        ISOLATION OF HBLV FROM PERIPHERAL BLOOD                                        LYMPHOCYTES OF PATIENTS WITH LYMPHOMA AND                                     LYMPHADENOPATHY                                                                                 Serology*   HBLV                                           Patient                                                                             Description      HTLV    HBLV  Isolation**                              ______________________________________                                        1     RC 29 WM  AIDS       +III  1:80  +                                          KS                                                                            B cell                                                                        lymphoma                                                                    2 HA 57 WM OHS  1:40 +                                                          AILD                                                                        3 PD 40 WM Dermatopathic +II 1:80 +                                             lymphademo- and                                                               pathy; IVDA +III                                                              T8.sup.+  skin                                                                infiltrate                                                                  4 GS 17 BM T-cell ALL -- 1:160 +                                                (T-4.sup.+)                                                                 5 RW 66 BM Mycoses -- 1:80 +                                                    Fungoides                                                                     (T-4.sup.+)                                                                   Cutaneous T-                                                                  cell Lymphoma                                                               6 BD 35 BF Immunoblastic -- 1:80 +                                              Lymphoma                                                                  ______________________________________                                         *Serology was done by indirect immunofluorescence using as standard a         reference virus isolated from patient GS.                                     **PBL from patients were cultured as the primary source of virus. Virus       particles were transmitted to fresh human cord blood. Positive cultures       were identified by morphology IF, and EM.                                     ***Definitions: KS = Kaposi's sarcoma, AILD  angioimmunoblastic               lymphadenopathy, IVDA  intravenous drug abuser.                          

                                      TABLE 2                                     __________________________________________________________________________    Cross-Reactivity of Nonhuman Primate Sera                                               Virus Used to Infected Target Cells                                           HBLV    EBV       RSV                                                  No. Positive No. Positive No. Positive                                        No. Tested (VCA)/No. Tested No. Tested                                       Serum Sources (Percent Positive) (Percent Positive) (Percent Positive)      __________________________________________________________________________    Old World Primate                                                               Chimpanzee 0/5 (0) 5/5 (100%) 0/4 (0)                                         Gorilla 0/3 (0) 2/3 (66.6%) 0/3 (0)                                           Orangutan 0/2 (0) 1/2 (50%) 0/2 (0)                                           Baboons 0/3 (0) 3/3 (100%) 0/3 (0)                                            Stumptail 0/2 (0) 1/2 (50%) 0/2 (0)                                           Rhesus 0/9 (0) 6/9 (66.6%) 0/7 (0)                                            African Green 0/10 (0) 6/10 (60%) 0/10 (0)                                    New World Primates                                                            Squirrel monkeys 0/10 (0) 0/10 (0%) 8/10 (80)                                 Owl monkeys 0/6 (0) 0/6 (0%) 3/6 (50)                                         Marmosets (common) 0/6 (0) 0/6 (0) 0/6 (0)                                    Marmoset (cottontop) 0/3 (0) 0/3 (0) 1/3 (33.3)                             __________________________________________________________________________     ZHV14 or ZVB70 probe, antiHBLV antibodies or purified HBLV               

                                      TABLE 3                                     __________________________________________________________________________    Immunological Cross Reactivities of HBLV to Other Human and Nonhuman           Primates Herpesviruses Antibody Viruses Used to Infect Target Cells                                  HSV-I     Af. Gr.                                                                           Rhesus                                    Source HBLV EBV HCMV and II VZV HVS CMV CMV                                 __________________________________________________________________________    ERV Monoclonal Antibody                                                                    -   +  -   -   -  -  -   -                                         (VCA, EA, MA)                                                                 HCMV Monoclonal Antibody - - + - - - - -                                      (VCA and EA)                                                                  HSV I and II Monoclonal - - - + - - - -                                       Antibody (early and late                                                      antigens)                                                                     VZV Monoclonal Antibody - - - - + - - -                                       (late antigens)                                                               HVS Monoclonal Antibody - - - - - + - -                                       (late antigens)                                                               Af. Green Monkey - - - - - - + -                                              CMV (hyperimmune serum)                                                       Rhesus Monkey - - - - - - - +                                                 CMV (hyperimmune serum)                                                     __________________________________________________________________________

    ______________________________________                                        Morphologic comparison of HBLV with other herpes viruses                        Feature    HBLV      HSV*    HCMV (6)                                                                              EBV (7)                                ______________________________________                                        Diameter of                                                                            60-80 nm  50-70 nm  64.3 nm 48 nm                                      nucleoid                                                                      Diameter of 95-105 nm 95-110 nm 106.4 nm 80 nm                                capsid                                                                        Symmetry of Icosahedral Icosahedral Icosahedral Icosahedral                   capsid                                                                        No. of 162 162 162 162                                                        capsomeres in                                                                 capsid                                                                        Thickness of Dense, Often Dense, Variable,                                    tegument prominent, indistinct, prominent, 20 nm                               25-40 nm 20-40 nm 24.4 nm                                                    Diameter of 160-200 nm 150-200 nm 174 nm 120 nm                               enveloped                                                                     virion                                                                      ______________________________________                                         *HSV used for this comparison was prepared simultaneously and under           identical conditions as HBLV.                                            

                  TABLE 5                                                         ______________________________________                                        EcoR1                  BamH1                                                  Fragment  MW (kb)      Fragment MW (kb)                                       ______________________________________                                        A         20.0         A        40.0                                            B 17.0 B 30.0                                                                 C 16.0 C 23.1                                                                 D 10.5 D 13.5                                                                 E 8.0 E 11.8                                                                  F 7.7 F 10.9                                                                  G 7.4 G 8.5                                                                   H 6.6 H 6.5                                                                   I 6.3 I 6.2                                                                   J1, J2 5.9 J 5.95                                                             K 5.4 K 5.6                                                                   L 5.0 L 3.4                                                                   M 4.5 M 2.6                                                                   N 4.36 N 2.05                                                                 O 3.85 O 1.95                                                                 P 3.75  172.05                                                                Q 3.5                                                                         R 3.25                                                                        S 3.05                                                                        T 2.95                                                                        U 2.5                                                                         V 2.4                                                                         W 2.3                                                                         X 2.25                                                                        Y 2.1                                                                         Z 1.75                                                                        Z1 1.5                                                                        Z2 1.49                                                                       Z3 1.35                                                                        168.55                                                                     ______________________________________                                    

What is claimed is:
 1. An isolated antibody which specifically binds toan antigenic molecule from an isolated human herpes virus having themorphology of a human herpes virus and a double-stranded DNA genome ofabout 170 Kb, wherein genomic DNA from said isolated human herpes virushybridizes under stringent conditions with nucleic acid of molecularclone ZVH14 (ATCC Accession No. 40,247); and further wherein said firstnucleic acid does not hybridize under said stringent conditions with thenucleic acid of:(a) Epstein-Barr virus; (b) human cytomegalovirus (CMV);(c) Herpes Simplex virus (HSV); (d) Varicella-Zoster virus (VZV); or (e)Herpes virus saimiri.
 2. A method of detecting HHV-6 in a biologicalsample comprising the steps of:(a) contacting the biological sample withthe antibody of claim 1, under conditions such that the antibody willspecifically bind to a human herpes virus antigenic molecule present insaid biological sample whereby a complex is formed of antibody andantigenic molecule; and (b) detecting for the presence or absence of thecomplex.
 3. The method of claim 2, wherein said method comprises awestern blot.